Assay for transmissible spongiform encephalopathies

ABSTRACT

Provided is a novel method for detecting Transmissible Spongiform encephalopathies (TSE). The method comprises: selecting a body fluid sample from a subject to determine whether the subject has transmissible spongiform encephalopathy; and detecting antibodies that immune react with  Spiroplasma  proteins in the sample. The method detects antibodies in the individual sera samples that react with recombinant  Spiroplasma -specific Hsp60 by standard ELISA methodology. This method provides for rapid detection of TSE by determination of presence of antibodies in test samples showing associated Spiroplasma infection that has become consistent with presence of TSE. The method provides a mechanism to detect presence of TSE without invasive techniques currently needed to make diagnosis using brain tissues for prion detection. This method provides means of detection of TSE in the live patient without need for using necropsy tissues. This method provides the first method discovered that detects antibodies against TSE infection-specific proteins in serum or cerebrospinal fluid or other body fluid samples.

This application claims priority from U.S. Provisional Application Ser.No. 60/573,814 (“the '814 application”) filed May 24, 2004. The '814application is incorporated herein by reference.

This work was supported by NIH NINDS 5RO1NS044000-O3 to Frank O.Bastian.

BACKGROUND OF THE INVENTION

The present invention presents an assay for transmissible spongiformencephalopathies in humans and other animals.

Transmissible spongiform encephalopathies (“TSE”) are diseases that canaffect humans and other animals. It is characterized by spongydegeneration of the brain. The disease is known as scrapie in sheep andgoats. The condition is known as both bovine spongiform encephalopathyor mad cow disease in cattle. There exist human variants of the diseaseknown as Kuru, Creutzfeldt-Jakob Disease (“CJD”), fatal familialinsomnia, and Gerstmann-Strausler disease. Deer and elk are known tocontract a variant of the disease known as chronic wasting disease. TSEis seen in farmed mink, known as transmissible mink encephalopathy.

At a molecular level, the disease is characterized by deposition ofprion protein in brains from TSE-afflicted humans or animals. The prionprotein is a normal constituent of brain tissue. In individuals affectedby transmissible spongiform encephalopathies, there is conformationalchange in the protein making it resistant to denaturation by proteolyticenzymes. Since the prion proteins of affected individuals are resistantto most methods of denaturation, most assays for the diseased version ofthe protein seek to differentiate between the diseased and normalversion of the protein. Typical assays for the protein first treatsuspected brain tissue with proteolytic enzymes, then seek to identifythe prion proteins (usually by polyacrylamide gel electrophoresisfollowed by western blotting with an antibody specific for both types ofprion proteins). In unaffected individuals, there is often no proteinavailable for recognition by the antibodies during western blottingfollowing proteolytic digestion. Because of the ability of diseasedprion proteins to resist proteolytic denaturation, they are recognizedby the anti-prion antibodies. An alternate approach is to use antibodyrecognition of the prion protein by Enzyme-Linked ImmunoadSorbent Assay(“ELISA”) using antibodies tagged with enzymes or fluorescent molecules.In either case, a fluorescent or calorimetric signal can be used toconclude the testing. Abnormal Prion has not been detected in sera orcerebrospinal fluid derived from TSE-afflicted individuals.

Current methods of testing for the presence of transmissible spongiformencephalopathies are for the most part conducted post-mortem. This isbecause an analysis of the prion proteins of the individuals must bedone and this is accomplished, by an analysis of brain tissue or othertissues such as tonsil. Thus, such analyses are extremely invasivenegating applicability to preclinical diagnosis of TSE. These assays,while useful, suffer from their reliance on brain tissue to provide adiagnostic result. The use of brain material is not feasible forscreening. This is especially not suitable when the individual to betested is a living human or for the purpose only to determine if theindividual is a safe blood donor. Therefore there exists a need todevelop a test that can provide diagnosis in a living individual withminimal invasion, preferably using sera samples.

An alternate approach has led to a better understanding of thepathogenesis of TSE whereby a wall-less bacterium called Spiroplasma isclosely associated with these diseases. The presence of Spiroplasma wasinitially discovered in an ultrastructural study of a brain biopsyobtained from a 46 year old CJD patient. Since then, the presence ofSpiroplasma genes in TSE brain tissues has been shown using moleculartechniques including polymerase chain reaction (PCR), Southern blottingand DNA sequence analyses. More recently, a unique Spiroplasma speciesfrom TSE-infected brain tissues has been isolated by passage throughembryonated eggs into cell-free media. The role of Spiroplasma infectionin the pathogenesis of TSE is supported by recent studies that haveshown the normal prion isoform to serve as a receptor protein for abacterium. It is presumed that interaction of Spiroplasma with the prionresults in the disease and accumulation of the misfolded prion protein.

The occurrence of a consistent Spiroplasma infection in association withindividual TSE cases provided the opportunity to develop a serum testfor the disease based upon the presence of antibodies generated againstSpiroplasma proteins. Heat shock protein 60 (Hsp60) was chosen becauseof prior data that showed interaction of bacterial-specific Hsp60protein on the bacterial surface with the prion receptor. It isnoteworthy that Hsp60 is widespread in nature. However, the Hsp60 ofbacteria is specific in that there is 70% homology with other bacteria,but a 100% homology among strains of the same genus. There is 50%homology of bacterial Hsp60 with human Hsp60 proteins. The inventor hasisolated the Hsp60 gene specific for Spiroplasma, produced recombinantSpiroplasma-specific Hsp60 recombinant protein and shown reactivity withsera from individual TSE cases by using ELISA.

The current methods of testing are also inefficient when applied tolarge numbers of livestock. Brain or neural material must be taken afterslaughter and processed to be assayed. This delay can result in thecarcass of the animal being placed into the human or animal food supplybefore testing can be concluded. Also, these postmortem assays cannot beused to test and produce groups of animals that are free oftransmissible spongiform encephalopathies or insure that animals withtransmissible spongiform encephalopathy do not co-mingle with diseasefree animals. The use of brain tissue, in addition to mandating apostmortem test, is also very inconvenient. An assay using a morereadily available bodily fluid such as sera or tissue offers a moreconvenient approach to testing of any animal type for TSE.

SUMMARY OF THE INVENTION

The present invention relates to methods of detecting transmissiblespongiform encephalopathies in animals, including human beings. Theinvention relies on the use of a protein of the Spiroplasma bacterium toidentify individuals affected with transmissible spongiformencephalopathies. Specifically, this invention uses the heat shockprotein 60 (Hsp60) of Spiroplasma mirum. Alternatively, other proteinsof Spiroplasma mirum could be used in place of Hsp60 in this assay.Additionally, the proteins of other members of the genus Spiroplasmacould be used to create the assay. Artificially synthesized peptidesrepresenting amino acid fragments of proteins from the genus Spiroplasmamay also be used.

The present invention uses the serum of an animal or human being as itstesting material. In another embodiment of the invention, whole bloodmay be used as the testing material. In yet another embodiment of thepresent invention, cerebrospinal fluid of an animal or human being mayserve as the testing material. Other body fluids such as urine, tearsand saliva may also serve as a testing material.

It is an object of the invention to provide a method of detecting atransmissible spongiform encephalopathy (TSE) disease in a mammal,including a human, a cow and a sheep, comprising detecting the presenceof an antibody to a protein from a bacterium of the genus Spiroplasma inthe serum of the mammal.

It is an object of this invention to provide a method of detecting atransmissible spongiform encephalopathy (TSE) disease in a mammal,including a human, a cow and a sheep, comprising contacting a serumsample from a mammal with at least a portion of heat shock protein 60(Hsp60) from a Spiroplasma bacterium and detecting a reaction product ofHsp60 or a portion thereof with a component of the plasma.

It is a further object of this invention to provide a method ofdiagnosing a TSE disease in a mammal comprising detecting the presenceof a Spiroplasma infection in a mammal.

It is a further object of this invention to provide a method ofdiagnosing a TSE disease in a mammal comprising detecting an antibody toSpiroplasma Hsp60 in a serum sample from a mammal.

It is a further object of this invention to provide a method ofdiagnosing a TSE disease in a mammal comprising detecting an antibody toSpiroplasma Hsp60 in a cerebrospinal fluid sample from a mammal.

It is a further object of this invention to provide a method ofdiagnosing a TSE disease in a mammal comprising detecting an antibody toSpiroplasma Hsp60 in a whole blood sample from a mammal.

It is a further object of this invention to provide an isolated Hsp60protein and an isolated polynucleotide encoding an Hsp60 protein havingDNA and amino acid sequences of SEQ ID NO. 1, and SEQ ID NO. 2,respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustrative gel electrophoresis of polymerase chainreaction (PCR) generated amplified products obtained by probing TSE andnormal brain tissues using oligonucleotide primers specific forSpiroplasma Hsp60 gene. The PCR amplified Spiroplasma Hsp60 gene productis only present in the TSE infected brain but not in normal brain orwater control.

FIG. 2 is an illustrative acrylamide electrophoresis of purifiedrecombinant Spiroplasma Hsp60 protein. The 45 kDa GST-tagged recombinantSpiroplasma Hsp60 protein eluted from the glutathione column is solubleand in sufficiently pure form (lane 2) for development of an indirectELISA.

FIG. 3 is a statistical analysis variance table (absorbance at 490 nm ofthe indirect ELISA plates) showing significant evidence of circulatingantibodies against Spiroplasma recombinant Hsp60 protein(p-value<0.0001) in sera from CJD patients.

FIG. 4 is a statistical analysis variance table (absorbance at 490 nm ofthe indirect ELISA plates) showing significant evidence of circulatingantibodies against Spiroplasma recombinant Hsp60 protein(p-value<0.0001) in sera from scrapie-infected sheep.

DETAILED DESCRIPTION OF THE INVENTION

The gene for heat shock protein 60 (Hsp60) of Spiroplasma mirum (GenBankID M24662) was identified by polymerase chain reaction (PCR) and DNAsequence analysis. The gene was cloned in a Topo vector (Invitrogen).The DNA and predicted amino acid sequence of Spiroplasma mirum Hsp60 isnot in the GenBank, has not been published, and is claimed here as partof this invention as described in SEQ ID NO 1 and SEQ ID NO 2. Newoligonucleotide primers were designed from this novel sequence and usedto probe DNA extracts from TSE brain tissues. As depicted in FIG. 1,which has a series of lanes 10 marked 1 through 7, twoSpiroplasma-specific Hsp60 probes revealed presence of amplified geneproducts in TSE brain tissues but not in normal brain tissues or a watercontrol. FIG. 1 shows presence of 130 and 140 bp amplified PCR productsin TSE-infected brains and not in controls. Of the lanes 10, the lanemarked 1 shows 100 bp markers. The lanes marked 2 and 5 show watercontrols. The lanes marked 3 and 6 show normal brain and the lanesmarked 4 and 7 show TSE-infected brain. The amplified PCR products arethe contrasting bands in the lanes marked 4 and 7. Other non-specificbanding represents primer/dimer.

The nucleic acid sequence encoding Spiroplasma mirum Hsp60 was placedinto a suitable bacterial expression system using GST fusion technology(Novagen). Lysates of bacteria expressing GST bound Hsp60 were used toisolate recombinant Hsp60 of Spiroplasma mirum purified by passingthrough a glutathione column. The recombinant Hsp60 was eluted from thecolumn as a soluble fraction. FIG. 2, which has lanes 11 marked 1, 2 and3, shows purified soluble fractions of recombinant Hsp60 of 45 kDa size.FIG. 2 shows elution fractions of GST-bound Spiroplasma Hsp60 in lanes11 marked 2 and 3 following passage over a glutathione column. The lanemarked 1 shows markers, and the lanes marked 2 and 3, show soluble 45kDa recombinant Spiroplasma Hsp60 protein.

Purified Spiroplasma mirum recombinant Hsp60 was attached to the surfaceof microwell plates. The wells of the plates were blocked with acommercially available blocking buffer. Serum samples from individualpatients who were Creutzfeldt-Jakob Disease positive or controls wereadded to the wells containing the Hsp60 protein. Following an incubationperiod, the serum samples were removed and each well was washed threetimes with either tris buffered saline or phosphate buffered saline. Agoat anti-human antibody tagged with horseradish peroxidase was added tothe wells. Following an incubation period, the wells were washed. Acalorimetric substrate was added to the wells. In the presence ofhorseradish peroxidase, the substrate is oxidized to produce a coloredsubstrate. The amount of analyte produced can be determined by measuringthe absorbance of individual samples or wells at the wavelengthsproduced by the oxidized substrates. All studies were done in triplicatealong with three wells with no primary antibody for determiningbackground. An alternate approach would be to use a chemiluminescentsubstrate of horseradish peroxidase. In such a case, the amount of ananalyte can be measured by the relative light units produced.

Statistical analysis variance tables (absorbance at 490 nm of theindirect ELISA plates) showed significant evidence of circulatingantibodies against Spiroplasma recombinant Hsp60 protein(p-value<0.0001) in sera from CJD patients. FIG. 3 compares absorbancereadings at 490 nm of thirty individual CJD sera samples compared tothirty individual normal sera. Column 12 shows distribution of platereadings at 490 nm of thirty individual CJD sera showing mean 15 andstandard error range 16. Column 13 shows distribution of plate readingsat 490 nm of thirty individual normal human sera showing mean 17 andstandard error range 18. Column 14 shows plate readings 19 at 490 nm ofplate background (no primary antibody).

Statistical analysis variance tables (absorbance at 490 nm of theindirect ELISA plates) showed significant evidence of circulatingantibodies against Spiroplasma recombinant Hsp60 protein(p-value<0.0001) in sera from scrapie-infected sheep. FIG. 4 comparesabsorbance readings at 490 nm of ten individual scrapie sheep serasamples compared to forty individual normal sheep sera. Column 20 showsdistribution of plate readings at 490 nm of ten individual scrapie serashowing mean 23 and standard error range 24. Column 21 showsdistribution of plate readings at 490 nm of forty individual normalsheep sera showing mean 25 and standard error range 26. Column 22 showsplate readings 27 at 490 nm of plate background (no primary antibody).

The statistical analyses of the ELISA results from the study of thirtyindividual CJD sera show that there is a >95% confidence in identifyingan individual CJD case and a >95% confidence in identifying anindividual normal control. Similarly, the study of individual scrapiesera show a >95% confidence in identifying an individualscrapie-infected animal and a >95% confidence in identifying anindividual normal control animal.

1. A method of detecting a transmissible spongiform encephalopathy (TSE)disease in a mammal comprising detecting the presence of an antibody toheat shock protein 60 (Hsp60) from a bacterium of the genus Spiroplasmain a body fluid sample from the mammal.
 2. A method as in claim 1,comprising contacting a serum or plasma sample from a mammal with Hsp60protein or an antigenic portion of Hsp60 protein from a Spiroplasmabacterium and detecting a reaction product of Hsp60 or an antigenicportion thereof with an antibody from the sample.
 3. A method as inclaim 1, wherein the sample comprises a serum or plasma sample from themammal.
 4. A method as in claim 3, comprising the sequential steps of(a) providing Spiroplasma Hsp60 protein or an antigenic portion of Hsp60protein, bound to a solid substrate to form a diagnostic substrate; (b)contacting the diagnostic substrate with serum or plasma from a mammalto bind to the diagnostic substrate a primary antibody specific forHsp60 present in the serum or plasma; (c) washing the diagnosticsubstrate to substantially remove proteins from the serum or plasma thatare not bound to the diagnostic substrate; (d) contacting the diagnosticsubstrate with a secondary antibody specific for the primary antibody ofthe mammal but not specific for Hsp60 protein to bind the secondaryantibody to at least a portion of any primary antibody bound to thediagnostic substrate; (e) washing the diagnostic substrate tosubstantially remove non-bound secondary antibody; and (f) measuring theamount of secondary antibody bound to the diagnostic substrate relativeto a standard.
 5. A method according to claim 1 wherein the mammal is ahuman.
 6. A method according to claim 2 wherein the mammal is a human.7. A method according to claim 3 wherein the mammal is a human.
 8. Amethod according to claim 4 wherein the mammal is a human.
 9. A methodaccording to claim 1 wherein the mammal is a cow.
 10. A method accordingto claim 2 wherein the mammal is a cow.
 11. A method according to claim3 wherein the mammal is a cow.
 12. A method according to claim 4 whereinthe mammal is a cow.
 13. A method according to claim 1 wherein themammal is a sheep.
 14. A method according to claim 2 wherein the mammalis a sheep.
 15. A method according to claim 3 wherein the mammal is asheep.
 16. A method according to claim 4 wherein the mammal is a sheep.17. A method as in claim 1, wherein the sample comprises a cerebrospinalfluid sample from the mammal.
 18. A method as in claim 17, comprisingcontacting a cerebrospinal fluid sample from a mammal with SpiroplasmaHsp60 protein or an antigenic portion of Hsp60 protein, and detecting areaction product of Hsp60 or an antigenic portion thereof with anantibody from the sample.
 19. A method as in claim 18, comprising thesequential steps of (a) providing Spiroplasma Hsp60 protein or anantigenic portion of Hsp60 protein, bound to a solid substrate to form adiagnostic substrate; (b) contacting the diagnostic substrate withcerebrospinal fluid from a mammal to bind to the diagnostic substrate aprimary antibody specific for Hsp60 present in the cerebrospinal fluid;(c) washing the diagnostic substrate to substantially remove proteinsfrom the cerebrospinal fluid that are not bound to the diagnosticsubstrate; (d) contacting the diagnostic substrate with a secondaryantibody specific for the primary antibody of the mammal but notspecific for Hsp60 protein to bind the secondary antibody to at least aportion of any primary antibody bound to the diagnostic substrate; (e)washing the diagnostic substrate to substantially remove non-boundsecondary antibody; and (f) measuring the amount of secondary antibodybound to the diagnostic substrate relative to a standard.
 20. A methodaccording to claim 17 wherein the mammal is a human.
 21. A methodaccording to claim 18 wherein the mammal is a human.
 22. A methodaccording to claim 19 wherein the mammal is a human.
 23. A methodaccording to claim 17 wherein the mammal is a cow.
 24. A methodaccording to claim 18 wherein the mammal is a cow.
 25. A methodaccording to claim 19 wherein the mammal is a cow.
 26. A methodaccording to claim 17 wherein the mammal is a sheep.
 27. A methodaccording to claim 18 wherein the mammal is a sheep.
 28. A methodaccording to claim 19 wherein the mammal is a sheep.
 29. An isolatedHsp60 protein having the amino acid residue sequence of SEQ ID NO: 2.30. An isolated polynucleotide encoding an Hsp60 protein having theamino acid residue sequence of SEQ ID NO:
 2. 31. An isolatedpolynucleotide having the nucleotide sequence of SEQ ID NO: 1.